EP2346999B1 - Use of sucrose as substrate for fermentative production of 1,2-propanediol - Google Patents

Use of sucrose as substrate for fermentative production of 1,2-propanediol Download PDF

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EP2346999B1
EP2346999B1 EP08875291.0A EP08875291A EP2346999B1 EP 2346999 B1 EP2346999 B1 EP 2346999B1 EP 08875291 A EP08875291 A EP 08875291A EP 2346999 B1 EP2346999 B1 EP 2346999B1
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sucrose
source
propanediol
microorganism
juice
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EP2346999A1 (en
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François VOELKER
Rainer Figge
Philippe Soucaille
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Metabolic Explorer SA
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Metabolic Explorer SA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric

Definitions

  • the present invention relates to fermentation processes .
  • this invention is related to the production of 1,2-propanediol by fermentation, from a sucrose-containing medium, in particular from plant biomass. whereby the microorganism is genetically modified by the introduction of the genes serKYABR or cscBKAR.
  • 1,2-propanediol or propylene glycol a C3 dialcohol
  • Propylene glycol has been increasingly used since 1993-1994 as a replacement for ethylene derivatives, which are recognised as being more toxic than propylene derivatives.
  • 1,2-propanediol is currently produced by chemical means using a propylene oxide hydration process that consumes large amounts of water.
  • Propylene oxide can be produced by either of two processes, one using epichlorhydrin, and the other hydroperoxide. Both routes use highly toxic substances.
  • the hydroperoxide route generates by-products such as tert- butanol and 1-phenyl ethanol. For the production of propylene to be profitable, a use must be found for these by-products.
  • the chemical route generally produces racemic 1,2-propanediol, whereas each of the two stereoisomers (R)1,2-propanediol and ( S )1,2-propanediol are of interest for certain applications (e.g. chiral starting materials for specialty chemicals and pharmaceutical products).
  • Carbon sources used in fermentation media generally consisted in carbohydrates, mostly derived from plants.
  • Sucrose is obtained from sugar plants such as sugar beet, sugarcane, sweet sorghum, sugar maple, sugar palms or blue agaves.
  • Starch is the most abundant storage carbohydrate in plants. The most important starch sources are cereals (corn, wheat, rice), manioc, sweet potatoes and potatoes. Starch is not metabolized by most microorgansims but can be processed to fermentable feedstocks by the starch industry.
  • Inulin or inulin-like polymers are the second most abundant storage carbohydrate in plants and are found in chicory, Jerusalem artichoke or dahlia.
  • Lignocellulosic biomass composed of cellulose, hemicellulose and lignin is also a promising source of carbohydrate but still under development (Peters, 2006).
  • the cost of the biotechnologically produced commodity chemicals are mainly related to the cost of raw material (i.e. the cost of the fermentation substrate)
  • use of refined sugars such as glucose or sucrose is not an economically sustainable choice for industrial scale production.
  • Less expensive substrates are needed that retain a high content of fermentable sugar.
  • sucrose containing carbon sources coming from the sugar industry represent a good option.
  • Sucrose is produced from sugar beet containing 16 to 24% sucrose by sugar beet processing in several steps.
  • the cleaned and washed beets are sliced into long strips called cossettes that are extracted with hot water by diffusion to get a sucrose juice called raw juice and containing 10 to 15% sucrose.
  • the second step is the purification of the raw juice by alkalization and carbonation using lime and then carbon dioxide to remove the impurities and get the thin juice.
  • the evaporation process increases the sucrose concentration in the thin juice by removing water to get the thick juice with a sucrose content of 50 to 65%. This concentrated sucrose juice is then crystallised and the crystals are separated by centrifugation and then washed and dried to get pure sugar.
  • One or more crystallisation steps can be applied to get sucrose of various purity grades.
  • By-products of sugar beet processing include pulp (the exhausted cossettes) and molasses (the remaining mother-liquor from the crystallisation having still a sucrose content of 40 to 60%).
  • Sucrose is also produced from sugar cane (7 to 20% sucrose content) by the sugar industry.
  • the harvested sugar cane is cleaned before the milling process for extraction of the juice.
  • the structure of the cane is broken and then grinded and at the same time the sucrose is extracted with water to get the raw juice.
  • the crushed cane exhausted from sugar is called bagasse.
  • This residue is primarily used as fuel source to generate process steam.
  • the raw juice is then clarified by adding lime and heating and the clarified juice is separated from the precipitate.
  • the lasts steps of the process, evaporation to get a concentrated syrup and crystallisation are essentially the same as for the sugar beet processing.
  • the by-products of sugarcane processing include bagasse, filter cake from clarification of raw juice and different kind of molasses, still containing significant amount of sucrose.
  • sucrose containing intermediates, products or by-products from the sugar processes may serve as fermentation feedstock.
  • sugar industry in Brazil is using the clarified sugarcane juice for ethanol production in order to use it as a substitute to gasoline.
  • Recent examples in literature using crude sucrose containing products include ethanol production from sugar beet diffusion juice by Zymomonas mobilis (Beckers et al., 1999), production of D-lactate from molasses by E . coli (Shukla et al., 2004) and production of D-lactate from sugarcane molasses, sugarcane juice or sugar beet juice by Lactobacillus delbrueckii (Calabia et al., 2007).
  • sucrose-positive bacteria i.e. bacteria able to utilize sucrose
  • the first one is based on a phosphoenol pyruvate (PEP)-dependent sucrose phosphotransferase system (sucrose PTS) where sucrose is taken up and phosphorylated using phosphoenol pyruvate (PEP) as a donor to yield intracellular sucrose-6-phosphate.
  • Sucrose-6-phosphate is then hydrolysed to D-glucose-6-phosphate and D-fructose by an invertase.
  • D-fructose is further phosphorylated to D-fructose-6-phosphate by an ATP-dependent fructokinase and can then enter the central metabolism.
  • a conjugative plasmid pUR400 bearing the genes scr KYABR coding for the sucrose PTS has been isolated from Salmonella (Schmid et al., 1982, Schmid et al., 1988).
  • Escherichia coli K12 and its derivatives cannot utilize sucrose. However, this ability can be conferred by the transfer of the genes coding for the two previously described systems. This has been demonstrated by transferring the plasmid pUR400 in E. coli K12 (Schmid et al, 1982) or different plasmids (including pKJL101-1) bearing the csc BKAR genes in a sucrose negative strain of E. coli (Robeis et al., 2002). As for industrial application, tryptophan production from sucrose has been documented in E . coli K12 (Tsunekawa et al., 1992), hydrogen production was shown in E. coli carrying the pUR400 plasmid (Penfold and Macaskie, 2004) and production of different amino-acids by transferring both systems, PTS and non-PTS was reported in patent application EP1149911 .
  • thermosaccharolyticum (later renamed T. thermosaccharolyticum ) by Cameron and Cooney (1986) but only traces were recorded whereas amount higher than 3 g/l with yields higher than 0,1 g/g substrate were obtained with other carbon sources.
  • sucrose-containing medium such as a juice or molasses from a plant feedstock, could be used as a substrate for the fermentative production of 1,2-propanediol.
  • the present invention is relative to a method for producing 1,2-propanediol by fermentation, comprising :
  • sucrose is obtained from biomass, in particular from plant biomass.
  • the terms 'cultivating', 'culture', 'growth' and 'fermentation' are used interchangeably to denote the growth of bacteria in an appropriate growth medium containing a simple carbon source. Fermentation is a classical process that can be performed under aerobic, microaerobic or anaerobic conditions.
  • the term 'appropriate medium' denotes a medium of known molecular composition adapted to the growth of the micro-organism.
  • said medium contains at least a source of phosphorus and a source of nitrogen.
  • Said appropriate medium is for example a mineral culture medium of known set composition adapted to the bacteria used, containing at least one carbon source.
  • Said appropriate medium may also designate any liquid comprising a source of nitrogen and/or a source of phosphorus, said liquid being added and/or mixed to the source of sucrose.
  • the mineral growth medium for Enterobacteriaceae can thus be of identical or similar composition to M9 medium (Anderson, 1946), M63 medium (Miller, 1992) or a medium such as defined by Schaefer et al.
  • the pH of the medium is adjusted to 6.8 with sodium hydroxide.
  • the carbon source 'glucose' can be replaced in this medium by any other carbon source, in particular by sucrose or any sucrose-containing carbon source such as sugarcane juice or sugar beet juice.
  • the growth medium for Clostridiaceae can be of identical or similar composition to Clostridial Growth Medium (CGM, Wiesenborn et al., 1987) or a mineral growth medium as given by Monot et al. (1982) or Vasconcelos et al. (1994).
  • 'sucrose' designates a disaccharide of glucose and fructose linked by a ⁇ (1,2) glycosidic bond, with the molecular formula G 12 H 22 O 11 . Its systematic name is ⁇ -D-glucopyranosyl- (1 ⁇ 2)- ⁇ -D-fructofuranoside.
  • sucrose source' or 'source of sucrose' designates any medium, liquid or solid, containing sucrose in different concentrations, in particular from 1 to 100% of sucrose.
  • producing 1,2-propanediol means that the production of the microorganism in the culture broth can be recorded unambiguously by standard analytical means known by those skilled in the art.
  • the limit of quantification of HPLC for 1,2-propanediol which is the preferred technique used to quantify this compound, is 25 mg/l. Therefore, the term "producing 1,2-propanediol" means according to the invention that the production levels have to be above 25 mg/l.
  • the term 'able to utilize sucrose as sole carbon source' indicates that the microorganism can grow in a medium containing sucrose as unique carbon source. Therefore, the definition of a "microorganism able to utilize sucrose as sole carbon source for the production of 1,2-propanediol" means that the microorganism, when grown in a medium containing sucrose as sole carbon source, can produce at least 25 mg/l of 1,2-propanediol.
  • the sucrose source in the culture medium can comprise additional carbon sources in addition to sucrose such as hexoses (such as glucose, galactose or lactose), pentoses, monosaccharides, disaccharides (such as sucrose, cellobiose or maltose)), oligosaccharides, starch or its derivatives, hemicelluloses, glycerol and combinations thereof.
  • hexoses such as glucose, galactose or lactose
  • pentoses monosaccharides, disaccharides (such as sucrose, cellobiose or maltose)), oligosaccharides, starch or its derivatives, hemicelluloses, glycerol and combinations thereof.
  • the microorganism has been genetically modified to be able to utilize sucrose as sole carbon source, for the production of 1,2-propanediol.
  • the microorganism comprises functional genes coding for a PTS sucrose utilization system.
  • a PTS sucrose utilization system is a system for sucrose utilization based on the transport of sucrose by a phosphoenolpyruvate (PEP)-dependent sucrose phosphotransferase system (Sucrose-PTS).
  • PEP phosphoenolpyruvate
  • a phosphotransferase system couples the transport of a sugar (e.g. sucrose or glucose) with the phosphorylation of the sugar using PEP as phosphate donor.
  • the sucrose-phosphate is cleaved into glucose-6-phosphate and fructose by an invertase.
  • Fructose is then phosphorylated into fructose-6-phosphate by a fructokinase.
  • the genes coding for this PTS sucrose utilization system can be controlled by a regulatory protein.
  • the microorganism expresses naturally or has been modified with the introduction of the genes : scr KYABR ( scr K coding for a fructokinase, scr Y coding for a porin, scrA coding for the Protein IIBC, scrB coding for a sucrose-6-P invertase, scr R coding for a repressor) from Salmonella.
  • a conjugative plasmid pUR400 bearing said genes scr -KYABR might be used to transform the microorganism.
  • These genes can be used all together in combination, or in any combination comprising at least one of these genes.
  • the gene scr R can be omitted.
  • genes has a more general meaning according to the invention, and covers the corresponding genes in other micro-organisms.
  • GenBank references of the genes from Salmonella those skilled in the art can determine equivalent genes in other organisms than Salmonella.
  • the means of identification of the homologous sequences and their percentage homologies are well-known to those skilled in the art, and include in particular the BLAST programmes that can be used on the website http://www.ncbi.nlm.nih.gov/BLAST/ with the default parameters indicated on that website.
  • the sequences obtained can be exploited (aligned) using for example the programmes CLUSTALW (http://www.ebi.ac.uk/clustalw/), with the default parameters indicated on these websites.
  • the PFAM database protein families database of alignments and hidden Markov models http://www.sanger.ac.uk/Software/Pfam/) is a large collection of alignments of protein sequences. Each PFAM makes it possible to visualise multiple alignments, view protein domains, evaluate distributions among organisms, gain access to other databases and visualise known protein structures.
  • COGs clusters of ortholagous groups of proteins http://www.ncbi.nlm.nih.gov/COG/
  • COGs are obtained by comparing protein sequences derived from 66 fully sequenced unicellular genomes representing 14 major phylogenetic lines.
  • Each COG is defined from at least three lines, making it possible to identify ancient conserved domains.
  • the microorganism comprises functional genes coding for a non-PTS sucrose utilization system.
  • a non-PTS sucrose utilization system is a system for sucrose utilization based on transport of sucrose by a system independent of phosphoenolpyruvate. After transport into the cell, the sucrose: is cleaved into glucose and fructose by an invertase. Fructose is then phosphorylated into fructose-6-phosphate by a fructokinase and glucose is phosphorylated into glucose-6-phosphate by a glucokinase.
  • the genes coding for this non-PTS sucrose utilization system can be controlled by a regulatory protein.
  • the microorganism expresses naturally or has been modified with the introduction of the genes from E . coli EC3132 i.e. the genes csc BKAR coding for a sucrose:proton symport transport system ( csc B), a fructokinase ( csc K), an invertase ( csc A ) and a sucrose-specific repressor ( csc R).
  • csc B sucrose:proton symport transport system
  • csc K fructokinase
  • csc A invertase
  • csc R sucrose-specific repressor
  • genes has a more general meaning according to the invention, and covers the corresponding genes in other micro-organisms.
  • GenBank references of the genes from E. coli those skilled in the art can determine equivalent genes in other organisms than E. coli (see above).
  • microorganism of the invention characterized by an improved activity of the biosynthesis pathway of 1,2-propanediol.
  • Microorganisms optimized for the production of 1,2-propanediol have been extensively disclosed in patent applications WO 2005/073364 , WO 2008/116853 , WO 2008/116852 and WO 2008/116848 .
  • microorganisms for use in the invention are bacteria, yeast or fungi.
  • the microorganism is selected from the group consisting of Enterobacteriaceae, Bacillaceae, Clostridiaceae, Streptomycetaceae and Corynebacteriaceae.
  • the microorganism is selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Thermoanaerobacterium thermosaccharolyticum, Clostridium sphenoides or Saccharomyces cerevisiae.
  • bacteria are fermented at temperatures between 20°C and 55°C, preferably between 25°C and 40°C, and preferably at about 35°C for Clostridiaceae and at about 37°C for Enterobacteriaceae.
  • Fermentation is a classical process that can be performed under aerobic, microaerobic or anaerobic conditions.
  • Under aerobic conditions' means that oxygen is provided to the culture by dissolving the gas into the liquid phase. This could be obtained by (1) sparging oxygen containing gas (e.g. air) into the liquid phase or (2) shaking the vessel containing the culture medium in order to transfer the oxygen contained in the head space into the liquid phase.
  • oxygen containing gas e.g. air
  • Advantages of the fermentation under aerobic conditions instead of anaerobic conditions is that the presence of oxygen as an electron acceptor improves the capacity of the strain to produce more energy in form of ATP for cellular processes. Therefore the strain has its general metabolism improved.
  • Micro-aerobic conditions are defined as culture conditions wherein low percentages of oxygen (e.g. using a mixture of gas containing between 0.1 and 10% of oxygen, completed to 100% with nitrogen), is dissolved into the liquid phase.
  • Anaerobic conditions are defined as culture conditions wherein no oxygen is provided to the culture medium. Strictly anaerobic conditions are obtained by sparging an inert gas like nitrogen into the culture medium to remove traces of other gas. Nitrate can be used as an electron acceptor to improve ATP production by the strain and improve its metabolism.
  • the sucrose source is obtained from biomass, in particular from plant biomass.
  • biomass in particular from plant biomass.
  • the whole plant or any specific part of a plant can be used to prepare the raw material used as sucrose source.
  • the preparation can be based on any treatment known by those skilled in the art to extract sucrose from a sucrose-containing plant biomass.
  • the sucrose source is obtained from a plant chosen among the group consisting of : sugarcane, sugar beet, sweet sorghum, sugar maple, sugar palm and blue agave.
  • the source of sucrose may in particular be obtained from sugarcane or sugar beet.
  • sucrose source can be used according to the invention, such as a juice, a concentrated juice, a syrup, a clarified juice, molasses or crystallized sucrose.
  • a preferred form is the raw juice from sugar cane, directly extracted from the plant without any treatment. Briefly, the harvested sugar cane is cleaned before the milling process for extraction of the juice. The structure of the cane is broken and then grinded, and at the same time the sucrose is extracted with water to get the raw juice.
  • the raw juice may then be clarified by adding lime and heating and the clarified juice is separated from the precipitate. Concentrated syrup is obtained by evaporation.
  • the sucrose source may be a final product, an intermediate product or a by-product of the sugarcane or sugar beet industry.
  • an appropriate medium for the growth of microorganisms can be designed either by using the sucrose source alone, i.e. the appropriate medium consists of the source of sucrose, or by complementing the sucrose source with a source of phosphorus and/or a source of nitrogen.
  • the sucrose source comprises at least 7% of sucrose.
  • Example 1 Construction of two strains of E . coli producing 1,2-propanediol and able to utilize sucrose as sole carbon source :
  • the E. coli strain MG1655 lpd*, ⁇ tpiA, ⁇ pflAB, ⁇ adhE, ⁇ ldhA: : Cm, ⁇ gloA, ⁇ aldA, ⁇ aldB, ⁇ edd evolved in chemostat culture under anaerobic conditions and adapted for growth in minimal medium was obtained as described in WO 2008/116852 .
  • This strain was named evolved strain E. coli MG1655 lpd*, ⁇ tpiA, ⁇ pflAB, ⁇ adhE, ⁇ ldhA: : Cm, ⁇ gloA, ⁇ aldA, ⁇ aldB, ⁇ edd,
  • the chloramphenicol resistance cassette was eliminated in said evolved strain and the presence of the modifications previously built in the strain was checked as previously described in WO 2008/116852 .
  • Example 2 production of 1,2-propanediol with sucrose as sole carbon source :
  • the strains obtained as described in Example I and the strain without plasmid used as control were cultivated in an Erlenmeyer flask assay under aerobic conditions in minimal medium MML11PG1_100 (see composition above) with 20 g/l glucose or sucrose as sole carbon source. Glucose as carbon source was used as control.
  • the culture was carried out at 34°C and the pH was maintained by buffering the culture medium with MOPS.
  • 1,2-propanediol, and residual glucose or sucrose in the fermentation broth were analysed by HPLC and the yields of 1,2-propanediol over glucose or the yields of 1,2-propanediol over sucrose were calculated.
  • Table 2 production of 1,2-propanediol in minimal medium with glucose or sucrose as carbon source. Strain / Carbon source 1,2-propanediol titer (g/l) 1,2-propanediol yield (g/g carbon source) Evolved E.
  • n is the number of repetitions of the same experiment.
  • Example 3 production of 1,2-propanediol with sugarcane juice as carbon source :
  • the strains obtained as described in Example 1 were cultivated in an Erlenmeyer flask assay under aerobic conditions in minimal medium MML11PG1_100 with sugarcane juice (20 g/l sucrose equivalent) as carbon source.
  • the sugarcane juice used in this experiment was obtained from a sugar mill in the south-east asia area and was collected right after the clarification with lime of the raw juice
  • the culture was carried out at 34°C and the pH was maintained by buffering the culture medium with MOPS.
  • 1,2-propanediol, and residual sucrose, glucose and fructose in the fermentation broth were analysed by HPLC and the yield of 1,2-propanediol over the sum of carbon sources was calculated.
  • Table 3 production of 1,2-propanediol in minimal medium with sugarcane juice as sucrose source. Strain / Carbon source 1,2-propanediol titer (g/l) 1,2-propanediol yield (g/g carbon source) Evolved E.
  • n is the number of repetitions of the same experiment.
  • Example 4 production of 1,2-propanediol with sugarcane juice alone or supplemented with nutrients :
  • the strains obtained as described in Example I were cultivated in an Erlenmeyer flask assay under aerobic conditions in a medium containing diluted sugarcane juice (20 g/l sucrose equivalent) either without supplementation or supplemented with phosphate and ammonium ((NH 4 ) 2 HPO 4 2,5 g/l), iron (Fe Citrate, H 2 O 0,1 g/l) and thiamine (0,02 g/l).
  • phosphate and ammonium (NH 4 ) 2 HPO 4 2,5 g/l)
  • iron Fe Citrate, H 2 O 0,1 g/l
  • thiamine (0,02 g/l).
  • the culture was carried out at 34°C and the pH was maintained by buffering the culture medium with MOPS (40 g/l).
  • MOPS 40 g/l
  • 1,2-propanediol, and residual sucrose, glucose and fructose in the fermentation broth were analysed by HPLC and the yield of 1,2-propanediol over the sum of carbon sources was calculated.
  • Table 4 production of 1,2-propanediol in sugarcane juice without supplementation or in supplemented sugarcane juice.
  • Strain / Carbon source 1,2-propanediol titer (g/l) 1,2-propanediol yield (g/g carbon source) Evolved E.

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US20110217744A1 (en) 2011-09-08
BRPI0823256B1 (pt) 2018-08-07
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US20150159181A1 (en) 2015-06-11
BRPI0823256B8 (pt) 2020-05-26
MX2011004842A (es) 2011-05-30
AR074092A1 (es) 2010-12-22
EP2346999A1 (en) 2011-07-27
BRPI0823256A2 (pt) 2014-11-18
KR101395971B1 (ko) 2014-05-16
KR20110088546A (ko) 2011-08-03
CN102272316B (zh) 2014-06-11
WO2010051849A1 (en) 2010-05-14
CA2741427A1 (en) 2010-05-14
US9617567B2 (en) 2017-04-11
JP2012507992A (ja) 2012-04-05
CN102272316A (zh) 2011-12-07

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